The long-term objective of this project is to characterize the molecular mechanisms by which eukaryotic transcriptional repressors control gene expression. Transcriptional regulation underlies crucial aspects of many biological processes, including development, immune responses, pathological states such as cancer, and the physiology of homeostasis and adaptation. While transcriptional activation in eukaryotes is beginning to be understood in mechanistic terms, negative regulation of genes by repressors is less well understood. This study will use complementary biochemical and genetic approaches to determine the mechanism by which the Drosophila knirps repressor protein regulates transcription. Drosophila is a good model system for such studies because of the extensive genetics, the sophisticated tools available for generating transgenic animals, and the established systems for in vitro transcription. The knirps repressor, a member of the nuclear receptor superfamily of transcription factors, plays an important role in early embryogenesis. Initial work indicates that knirps can repress enhancers and basal promoters from a short range. This activity is characteristic of an important family of repressors in Drosophila, and likely represents a common mode of repression in other eukaryotes. We will define regions of knirps protein vital for repression in vivo using two novel Drosophila embryo assays. This genetic information will complement in vitro studies, including in vitro transcriptional assays, which will provide mechanistic insights into knirps activity, and direct binding studies, which will identify proteins which may interact with knirps to mediate repression. We will also identify knirps-interacting proteins using a yeast two-hybrid screen, and evaluate candidate target proteins using genetic and biochemical information developed from the other assays. It is clear that the basic circuitry regulating genes in Drosophila and vertebrates is highly conserved. Analysis of repression is crucial for understanding biological processes ranging from development to cancer. For instance, transcriptional repressors such as the retinoblastoma, Wilm's tumor, and MDM2 proteins are important factors in human cancers. This project will employ a multidisciplinary approach to provide detailed mechanistic information about this important form of gene regulation.